Bina Isaac

Neutrophil Infiltration and Oxidative Stress May Play a Critical Role in Methotrexate-Induced Renal Damage

Background: Nephrotoxicity is one of the adverse side effects of methotrexate (MTX) chemotherapy. The mechanism of renotoxicity of MTX is not fully understood. It is essential to understand the mechanism of nephrotoxicity of MTX in order to diminish the side effects and hence maximize the benefits of chemotherapy. Objectives: The aim of the study was to verify whether oxidative stress and neutrophil infiltration play a role in MTX-induced renal damage using a rat model. Methods: Adult male rats were administered MTX at the dose of 7 mg/kg body weight intraperitoneally for 3 consecutive days and sacrificed 12 or 24 h after the last dose. Vehicle-treated rats served as controls. The kidneys were removed and used for light microscopic and biochemical studies. Myeloperoxidase activity, a marker of neutrophil infiltration was measured in kidney homogenates along with the markers of oxidative damage including protein carbonyl content, protein thiol and malondialdehyde. The activities of the antioxidant enzymes, namely glutathione peroxidase, glutathione S-transferase, superoxide dismutase and catalase, were also assayed. Results: MTX treatment induced damage to the glomeruli and tubules. Plasma creatinine levels in the MTX-treated rats were significantly elevated compared with controls. A significant increase in myeloperoxidase activity (p < 0.05) was observed in the kidneys of MTX-treated rats. Protein carbonyl content and malondialdehyde, sensitive and reliable markers of oxidative damage to proteins and lipids, respectively, were significantly elevated (p < 0.01) in the kidneys of MTX-treated rats compared with controls. The activities of the antioxidant enzymes, namely, superoxide dismutase and glutathione peroxidase, were significantly elevated (p < 0.01 and p < 0.05, respectively) in kidneys of rats following MTX treatment. Conclusion: The results of the present study provide evidence for the role of neutrophil infiltration and oxidative stress in MTX-induced renal damage. Administration of inhibitors of myeloperoxidase or scavenging hypochlorous acid, the product of myeloperoxidase, by supplementation with antioxidants as an adjuvant therapy may be promising in alleviating the renal side effect of MTX.

Depletion of the cellular antioxidant system contributes to tenofovir disoproxil fumarate - induced mitochondrial damage and increased oxido-nitrosative stress in the kidney

BackgroundNephrotoxicity is a dose limiting side effect of tenofovir, a reverse transcriptase inhibitor that is used for the treatment of HIV infection. The mechanism of tenofovir nephrotoxicity is not clear. Tenofovir is specifically toxic to the proximal convoluted tubules and proximal tubular mitochondria are the targets of tenofovir cytotoxicity. Damaged mitochondria are major sources of reactive oxygen species and cellular damage is reported to occur after the antioxidants are depleted. The purpose of the study is to investigate the alterations in cellular antioxidant system in tenofovir induced renal damage using a rat model.ResultsChronic tenofovir administration to adult Wistar rats resulted in proximal tubular damage (as evidenced by light microscopy), proximal tubular dysfunction (as shown by Fanconi syndrome and tubular proteinuria), and extensive proximal tubular mitochondrial injury (as revealed by electron microscopy). A 50% increase in protein carbonyl content was observed in the kidneys of TDF treated rats as compared with the control. Reduced glutathione was decreased by 50%. The activity of superoxide dismutase was decreased by 57%, glutathione peroxidase by 45%, and glutathione reductase by 150% as compared with control. Carbonic Anhydrase activity was decreased by 45% in the TDF treated rat kidneys as compared with control. Succinate dehydrogenase activity, an indicator of mitochondrial activity was decreased by 29% in the TDF treated rat kidneys as compared with controls, suggesting mitochondrial dysfunction.ConclusionTenofovir- induced mitochondrial damage and increased oxidative stress in the rat kidneys may be due to depletion of the antioxidant system particularly, the glutathione dependent system and MnSOD.

Mitochondrial dysfunction and electron transport chain complex defect in a rat model of tenofovir disoproxil fumarate nephrotoxicity.

The long‐term use of tenofovir, a commonly used anti‐HIV drug, can result in renal damage. The mechanism of tenofovir disoproxil fumarate (TDF) nephrotoxicity is not clear, although it has been shown to target proximal tubular mitochondria. In the present study, the effects of chronic TDF treatment on the proximal tubular function, renal mitochondrial function, and the activities of the electron transport chain (ETC) complexes were studied in rats. Damage to proximal tubular mitochondria and proximal tubular dysfunction was observed. The impaired mitochondrial function such as the respiratory control ratio, 2‐(4,5‐dimethyl‐2‐thiazolyl)‐3,5‐diphenyl‐2H‐tetrazolium bromide (MTT) reduction, and mitochondrial swelling was observed. The activities of the electron chain complexes I, II, IV, and V were decreased by 46%, 20%, 26%, and 21%, respectively, in the TDF‐treated rat kidneys. It is suggested that TDF induced proximal tubular mitochondrial dysfunction and ETC defects may impair ATP production, resulting in proximal tubular damage and dysfunction.

Mitochondrial dysfunction and respiratory chain defects in a rodent model of methotrexate-induced enteritis

The efficacy of methotrexate (MTX), a widely used chemotherapeutic drug, is limited by its gastrointestinal toxicity and the mechanism of which is not clear. The present study investigates the possible role of mitochondrial damage in MTX-induced enteritis. Small intestinal injury was induced in Wistar rats by the administration of 7 mg kg−1 body wt. MTX intraperitoneally for 3 consecutive days. MTX administration resulted in severe small intestinal injury and extensive damage to enterocyte mitochondria. Respiratory control ratio, the single most useful and reliable test of mitochondrial function, and 3-(4,5-dimethylthiazol-2-yll)-2,5-diphenyltetrazolium bromide reduction, a measure of cell viability were significantly reduced in all the fractions of MTX-treated rat enterocytes. A massive decrease (nearly 70%) in the activities of complexes II and IV was also observed. The results of the present study suggest that MTX-induced damage to enterocyte mitochondria may play a critical role in enteritis. MTX-induced alteration in mitochondrial structure may cause its dysfunction and decreases the activities of the electron chain complexes. MTX-induced mitochondrial damage can result in reduced adenosine triphosphate synthesis, thereby interfering with nutrient absorption and enterocyte renewal. This derangement may contribute to malabsorption of nutrients, diarrhea, and weight loss seen in patients on MTX chemotherapy.

Ultrastructural changes in the rat kidney after single dose of cyclophosphamide—Possible roles for peroxisome proliferation and lysosomal dysfunction in cyclophosphamide-induced renal damage

Electron microscopy was used to examine changes in the subcellular organelles of the rat kidney at different time intervals after a single exposure to cyclophosphamide (CP). The morphological changes were studied at different time points (6 hrs, 16 hrs and 24 hrs) after a single-dose administration of CP. Six rats were killed at each time intervals after the administration of CP. Saline-treated rats served as controls. CP administration resulted in alterations in various subcellular organelles including peroxisomes, lysosomes, mitochondria, and the endoplasmic reticulum (ER) of the renal tubular epithelium as well as damage to the glomerulus. The basement membrane of the glomerulus was thickened. Many podocytes were destroyed. The nucleoplasm of the endothelial cell showed fewer granularities. The tubules were distorted and the brush border was destroyed. Two striking features in the renal tubular cells are increase in number and size of the peroxisomes (peroxisome proliferation) and decrease in the number of lysosomes. The mitochondria were elongated and the number was increased in the tubules of CP-treated rats. The ER was dilated. Cell necrosis was also seen. This study is an evidence of changes in morphology of rat kidney after induction of renal damage by a single dose of CP. Since transmission electron microscopy is the highest magnification tool at present, it can be useful in estimating the degree of injury and outcome of alternative treatment strategies in the management of CP-induced renal damage after establishing a scoring system.

The effects of oral glutamine on cyclophosphamide-induced nephrotoxicity in rats.

Nephrotoxicity is one of the adverse side effects of cyclophosphamide (CP) chemotherapy. In a recent study, we have demonstrated that oxidative stress and glutathione depletion play important roles in CP-induced renal damage. The aim of the study was to verify whether glutamine, the precursor for glutathione synthesis, prevents CP-induced oxidative stress and renal damage using a rat model. Adult male rats were administered a single dose of 150 mg/ kg body weight of CP intraperitoneally. The glutamine-pretreated rats were administered 1 gm/kg body weight of glutamine orally 2 h before the administration of CP. Vehicle/glutaminetreated rats served as controls. All the rats were killed 16 h after the dose of CP/vehicle. The kidneys were removed and used for light microscopic and biochemical studies. The markers of oxidative stress including malondialdehyde content, protein carbonyl content, protein thiol, reduced glutathione and myeloperoxidase activity, a marker of neutrophil infiltration, were measured in kidney homogenates. CP treatment-induced damage to kidney involved the glomeruli and the tubules. Pretreatment with glutamine reduced CP-induced glutathione depletion and increased myeloperoxidase activity. However, it did not prevent CP-induced lipid peroxidation, protein carbonylation and renal damage. The results of the present study suggest that glutamine pretreatment does not prevent CP-induced lipid peroxidation and renal damage, although it prevents CP-induced glutathione depletion and neutrophil infiltration significantly. It is suggested that mechanisms other than oxidative stress may also be involved and/or oxidative stress may be consequence and not the cause of CP induced renal damage.

Preclinical efficacy of melatonin in the amelioration of tenofovir nephrotoxicity by the attenuation of oxidative stress, nitrosative stress, and inflammation in rats.

Abstract Background: Nephrotoxicity is a dose-limiting side effect of long-term use of tenofovir, a reverse transcriptase inhibitor that is used for the treatment of human immunodeficiency virus (HIV) infection. Identifying an agent that prevents tenofovir disoproxil fumarate (TDF)-induced renal injury can lead to better tolerance to TDF, and a more effective treatment can be achieved in HIV infected patients. Recent studies show that oxidative stress, nitrosative stress, and inflammation play a role in TDF nephrotoxicity. The present study is aimed at investigating whether melatonin, a potent antioxidant and anti-inflammatory agent, protects against TDF nephrotoxicity in rats. Methods: Adult male rats were used for the study. Some rats received 600 mg/kg body weight TDF by gavage for 35 days, while others received once daily 20 mg/kg body weight melatonin i.p. 2 h before TDF administration. All the rats were sacrificed on the 36th day, after overnight fast. Results: Melatonin pretreatment protected the rats against TDF nephrotoxicity both histologically and biochemically. Biochemically, melatonin pretreatment attenuated TDF-induced renal oxidative stress, nitrosative stress, and inflammation and preserved proximal tubular function. Histologically, melatonin pretreatment prevented TDF-induced proximal tubular injury and mitochondrial injury such as swelling, disruption of cristae, and deposition of amorphous material in the matrix. It restored the lysosomal and mitochondrial numbers in the proximal tubules also. Conclusions: Melatonin pretreatment protects rats from tenofovir-induced damage to proximal tubular mitochondria by attenuating oxidative stress, nitrosative stress, and inflammation. This suggests that it may be useful in ameliorating TDF nephrotoxicity in humans.

NF-κB-iNOS-COX2-TNF α inflammatory signaling pathway plays an important role in methotrexate induced small intestinal injury in rats

Although methotrexate is widely used in clinics as an anticancer agent, its utility is limited by its gastrointestinal toxicity, the mechanism of which is unclear. The role of NFκB inflammatory pathway in MTX induced mucositis was investigated in the present study. GI injury was induced in adult Wistar rats by the administration of 3 consecutive i.p . injections of MTX. On the fourth day, the rats were sacrificed and the small intestine was removed; A piece was used for light microscopy, immunohistochemistry, immunofluorescence studies . The mucosa was collected and used for the analysis of protein and mRNA expressions of NFκB and its target genes by the western blot, RT-PCR respectively. MTX treatment resulted in NFκB activation and nuclear translocation as evidenced by immunofluorescence, immunohistochemistry , and western blot. NFκB mRNA was also increased. There was increased protein and mRNA expressions of NFκB target genes, TNF-α, iNOS, COX-2, PLA2, HO-1, HSP70, MMPs 2 and 9 . Aminoguanidine pretreatment (30mg/ 50mg /kg body wt.) attenuated MTX induced activation of NFκB and its proinflammatory target genes and improved MTX induced morphological changes. Aminoguanidine has protective effects against MTX induced gastrointestinal mucositis in rats.

Mitochondrial pathway of apoptosis and necrosis contribute to tenofovir disoproxil fumarate–induced renal damage in rats

Tenofovir disoproxil fumarate (TDF) is currently the only nucleotide analogue reverse-transcriptase inhibitor that is approved by the Food and Drug administration (FDA), USA, for the treatment of human immunodeficiency virus (HIV) infection. In recent days, renal toxicity is becoming common i HIV patients treated with TDF. However, the mechanism of tenofovir nephrotoxicity is not clear. We hypothesized that mitochondrial pathway of apoptosis, poly [ADP-ribose] polymerase (PARP) overactivation and neutrophil infiltration may contribute to tenofovir-induced renal damage. Renal damage was induced in adult male Wistar rats by the oral administration of 600 mg/kg body weight daily for five consecutive weeks. Kidneys were removed and used for histological and biochemical analyses. Apoptosis was detected by terminal deoxynucleotidyl transferase biotin–deoxyuridine triphosphate nick end-labelling (TUNEL) assay and caspase 3 activity and protein expression; mitochondrial pathway of apoptosis by cyt c release; and PARP activation by immunofluorescence, immunohistochemistry and Western blot techniques. Myeloperoxidase (MPO) activity was measured as a marker of neutrophil infiltration. TDF administration resulted in increased number of TUNEL-positive cells, activation of caspase 3 and release of cyt c from mitochondria into the cytosol in the kidneys. There was increased nuclear localization of PARP as well as increase in its protein level in the TDF-treated rat kidneys. In addition, renal MPO activity was increased ninefold as compared to controls. The results of the present study show that mitochondrial apoptotic pathway, PARP overactivation and neutrophil infiltration contribute to tenofovir-induced renal damage in rats.

Clinical anatomy of the inferior epigastric artery with special relevance to invasive procedures of the anterior abdominal wall.

Introduction: Injury to the inferior epigastric artery (IEA) has been reported following lower abdominal wall surgical incisions, abdominal peritoneocentesis and trocar placements at laparoscopic port sites, resulting in the formation of abdominal wall haematomas that may expand considerably due to lack of tissue resistance. The aim of this study was to localise its course in relation to standard anatomic landmarks and suggest safe areas for performance of invasive procedures. Materials and Methods: Sixty IEAs of 30 adult cadavers (male = 19; female = 11) were dissected and the course of the IEA noted in relation to the mid-inguinal point, anterior superior iliac spine (ASIS) and umbilicus. Results: The mean distance of the IEA from the midline was 4.45 ± 1.42 cm at the level of the mid-inguinal point, 4.10 ± 1.15 cm at the level of ASIS and 4.49 ± 1.15 cm at the level of umbilicus. There was an average of 3.3 branches per IEA with more branches arising from its lateral aspect. The IEA was situated within one-third (32%) of the distance between the midline and the sagittal plane through ASIS at all levels. Conclusion: To avoid injury to IEA, trocars can be safely inserted 5.5 cm [mean + 1 standard deviation (SD)] away from the midline (or) slightly more than one-third of the distance between the midline and a sagittal plane running through ASIS. These findings may be useful not only for laparoscopic procedures but also for image-guided biopsy, abdominal paracentesis, and placement of abdominal drains.